1. Field of the Technology
The disclosure relates to the field of optical methods for super-resolution imaging of live cells, specifically to an optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample which produces nanometer-resolved three dimensional images of very small and moving features in live cells and in a matter of seconds.
2. Description of the Prior Art
Several optical microscopy techniques have been previously developed to “break” the diffraction limit and to produce nanometer-resolved images. These techniques can be broadly classified as “physical” techniques in which ingenious approaches are used to break the diffraction limit. For example the STEO (stimulated emission depletion) technique uses stimulated emission to reduce the effective size of the PSF (point spread function). Other methods have used the determination of the center of mass of the fluorescence emission due to single molecules to obtain images with nanometer resolution of cellular features. The PALM and the STORM techniques and their variants also use this approach
Current methods in laser scanning confocal microscopy are based on moving a laser spot in a predetermined pattern, for example in a raster scan path, to obtain the intensity in each point of a plane of focus. These scanning techniques are inefficient however when the features to be imaged are at the nanoscale and sparse since the scanning path crosses the object to be imaged in only a few points. The efficiency of a predetermined scanning pattern, defined as the ratio of the time the laser beam is on a feature with respect to the total time of scanning, further decreases in three dimensions. Live cell structures like protrusions or microvilli are continually remodeled changing shape and position. Current imaging methods, including the super-resolution techniques known as STED and PALM, are inadequate to detect the dynamic of chemical reactions in these tiny three dimensional structures, which occur in the millisecond to second time scale.